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Adherent culture method of neural stem cells

A neural stem cell and adherent culture technology, applied in the field of in vitro stem cell culture, can solve the problems of reduced self-renewal ability of neural stem cells, uncontrollable overall growth state, and inability to quantify the number of cells, etc., to achieve shortened digestion time, low price, and low cost Effect

Inactive Publication Date: 2017-12-15
SHANDONG QILU STEM CELL ENG
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Although the instantaneous viability of the cells obtained by the combined digestion method reaches over 90% after digestion, the self-renewal ability of neural stem cells is reduced and a large number of cells continue to die. Statistics show that the cell proliferation rate of neural stem cell spheres after 7 days of digestion is only 6- 8%
Moreover, the neural stem cell spheroids obtained by the suspension culture method are often of different sizes, and the overall growth state is uncontrollable; if the diameter of the neural stem cell spheroid is too large, the cells in the inner layer of the spheroid will not be able to obtain enough nutrition and eventually die, which will greatly affect Cell harvest
With the promotion of cell therapy methods, the standards for stem cell preparations used in preclinical research have become more stringent, and the suspension culture of neural stem cells cannot quantify the number of cells in each neural stem cell sphere, so the amount of cells harvested after digestion cannot be guaranteed

Method used

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  • Adherent culture method of neural stem cells
  • Adherent culture method of neural stem cells
  • Adherent culture method of neural stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Neural stem cell culture.

[0036] Prepare primary culture medium, containing 500 mL of DMEM / F-12 medium, basic fibroblast growth factor (basic FGF) 40 ng / mL, epidermal growth factor (EGF) 40 ng / mL, BIT 9500 50 mL, Heparin (Heparin) 5 μg / mL, L-glutamine 2 mM, Antibiotic-Antimycotic mixture (1:100) 5 mL.

[0037] Prepare subculture medium, containing DMEM / F-12 medium 500 mL, basic FGF 20 ng / mL, EGF 20 ng / mL, BIT9500 50 mL, L-glutamine 2 mM, Antibiotic-Antimycotic mixture (1:100) 5 mL .

[0038] (1) Add the forebrain tissue cells to the primary culture medium of neural stem cells preheated to 37°C, mix well and count the total cell number by trypan blue staining; press 3×10 6 Individuals / mL were inoculated into T75 culture flasks, the culture system in each flask was 15 mL, and 4 flasks were cultured in total; the culture flasks were placed at 37°C, 5% CO 2 cultured in an incubator.

[0039] After 36 hours of primary culture, the non-neural cells died, and...

Embodiment 2

[0042] Example 2 Identification of neural stem cells.

[0043] Take the neural stem cells passed to the 15th generation in Example 1, digest them with Accutase, and press 5×10 5 One piece / slice was seeded on a 14 mm diameter cell slide coated with poly-lysine, and adhered to the wall overnight for indirect immunofluorescence identification of neural stem cell markers (Nestin, SOX2).

[0044] The neural stem cell slides were collected and placed in a six-well plate, rinsed once with PBS balanced salt solution; fixed with 4% paraformaldehyde solution at room temperature for 20 min, and then aspirated off the fixative solution; then added 0.1% TritonX-100 solution, and incubated at room temperature for 10 minutes After 1 min, all the liquid was sucked off, and the slices were rinsed with PBS balanced salt solution 5 times, 5 min each time; the primary antibody Anti-humanNestin / Anti-human SOX2 was diluted with 5% donkey serum to a final concentration of 1:400; the diluted Antibod...

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Abstract

The invention provides an adherent culture method of neural stem cells. The method includes the steps of conducting primary culture on forebrain histocytes to obtain a neural stem cell sphere suspension; digesting neural stem cell spheres into suspension neural stem cells, inoculating the suspension neural stem cells into a petri dish wrapped by human fibronectin for culture, and conducting continuous cell culture after the cells grow to a certain integration degree; using digestive enzymes to digest the cells during follow-up continuous cell culture, after the cells shrink, blowing down the cells and then conducting centrifugation to obtain cell sediments for continuous cell culture. The adherent culture method of the neural stem cells has the advantages that monolayer adherent culture of the cells is achieved in a limited culture area, the cell harvest yield per unit area is clear, and the cell quantity after harvest can be ensured; moreover, the cells after the adherent culture are easy to digest, damage to the cells is reduced, and the viability of the obtained cells is high; the used human fibronectin for facilitating the adherent culture of the neural stem cells is low in price and capable of being utilized over and over again, other reagents are all common, and the cost of the unit cell quantity is low.

Description

technical field [0001] The invention relates to a method for converting suspension-cultured neural stem cells into adherent culture, and belongs to the technical field of in vitro stem cell culture. Background technique [0002] Neural stem cells are a kind of stem cells with self-renewal and multi-lineage differentiation ability, which mainly exist in the embryonic and adult central nervous system. Neural stem cells can differentiate into various types of cells, such as neurons, astrocytes, oligodendrocytes, etc. During embryonic development, these cells participate in the construction of neural tissues. Small numbers of neural stem cells remain in the adult CNS, and these cells are thought to be involved in the repair of damaged nervous systems. So far, a large number of scholars have used neural stem cells cultured in vitro to study the molecular and cellular mechanisms of nervous system development. In addition, more and more reports believe that neural stem cell trans...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2500/32C12N2501/11C12N2501/115C12N2501/91C12N2533/52
Inventor 陈恒王帅田振辉宋振涛曲廷瑜
Owner SHANDONG QILU STEM CELL ENG
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